WO2015091770A1 - Efficacité et innocuité d'une souche de bacillus d'origine marine pour une utilisation en tant que probiotique intégré aux aliments pour des cochons récemment sevrés - Google Patents

Efficacité et innocuité d'une souche de bacillus d'origine marine pour une utilisation en tant que probiotique intégré aux aliments pour des cochons récemment sevrés Download PDF

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Publication number
WO2015091770A1
WO2015091770A1 PCT/EP2014/078428 EP2014078428W WO2015091770A1 WO 2015091770 A1 WO2015091770 A1 WO 2015091770A1 EP 2014078428 W EP2014078428 W EP 2014078428W WO 2015091770 A1 WO2015091770 A1 WO 2015091770A1
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medicated
treatment
pigs
coli
spore suspension
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PCT/EP2014/078428
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English (en)
Inventor
Gillian GARDINER
Peadar G. LAWLOR
Peter Mcloughlin
Helen HUGHES
Laurie O'SULLIVAN
Maria LUZ PRIETO
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Waterford Institute Of Technology
Teagasc, The Agriculture And Food Development Authority
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Publication of WO2015091770A1 publication Critical patent/WO2015091770A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms

Definitions

  • the present invention relates to the treatment of animals to improve their performance and health through the use of a Bacillus pumilus spore suspension.
  • the invention relates to the use of the probiotic Bacillus pumilus to decrease the bacteria within the animal which may adversely affect performance and health.
  • Weaning is a stressful event for young animals and in particular pigs which is characterized by gastrointestinal disturbances caused by physiological, immunological and microbiological changes within the gastrointestinal tract.
  • animals are highly susceptible to enteric diseases, and those caused by Escherichia coli (e.g. post-weaning diarrhea and edema disease) are responsible for considerable economic losses in the pig industry.
  • in-feed antibiotics have long been used for the elimination or reduction of pathogenic bacteria, in particular E. coli, during the post-weaning period.
  • the routine use of in-feed antibiotics was banned in the EU in 2006 (Regulation EC/1831/2003), although their use is still permitted under veterinary prescription as the need arises.
  • apramycin (Apralan G200, Elanco Animal Health, Eli Lilly & Co. Ltd) was prescribed for use on the pig farm where the current study was conducted to control persistent edema disease during the post-weaning period.
  • antibiotic -resistance is a major human health issue and effective alternatives to antibiotics are required.
  • In-feed zinc oxide at pharmacological concentrations (i.e. concentrations in excess of normal dietary requirements) is also commonly used for enteric disease prevention in weaned pigs but there are concerns about its accumulation in the environment.
  • Probiotics are defined as 'live microorganisms which when administered in adequate amounts confer a health benefit on the host'. They offer potential as an alternative to antibiotics for pigs, both as a means of controlling enteric pathogens and improving growth rate and feed conversion. Together with modulation of the immune system and competitive exclusion, antimicrobial production is one of the suggested mechanisms of action of probiotics. The latter can therefore be considered a probiotic trait and is often listed as one of the properties required of a strain for it to be considered probiotic. This is backed up by the fact that strains selected in vitro for their anti-E. coli activity, have proven successful in reducing E. coli shedding, preventing diarrhea and improving growth performance in pigs.
  • the aim of the trial was to evaluate this pre- screened Bacillus strain for use as an in-feed probiotic for newly weaned pigs in comparison to a negative control treatment without antibiotic or pharmacological levels of zinc oxide (non-medicated treatment) and a positive control treatment containing apramycin and pharmacological levels of zinc oxide (medicated treatment).
  • Key parameters including growth performance and health indicators were investigated in order to evaluate safety and efficacy in vivo.
  • the B. pumilus treatment decreased ileal E. coli counts in a manner similar to medicated treatment but without the adverse effects on growth performance, Lactobacillus counts, cecal short chain fatty acid concentration and possible liver toxicity experienced with the medicated treatment.
  • Treatments 16 were: (1) non-medicated diet; (2) medicated diet with apramycin (200 mg/kg) and pharmacological levels of zinc oxide (2,500 mg zinc/kg) and (3) B. pumilus diet (non-medicated diet + 10 10 spores/day B. pumilus).
  • pumilus treatment had lower overall lymphocyte and higher granulocyte percentages (P ⁇ 0.001) and higher numbers of jejunal goblet cells (P ⁇ 0.01) than pigs on either of the other two treatments or the non-medicated treatment, respectively.
  • P ⁇ 0.001 lymphocyte and higher granulocyte percentages
  • P ⁇ 0.01 numbers of jejunal goblet cells
  • Bacillus pumilus strain spore suspension for use as an in-feed probiotic in animals where the suspension satisfy a number of probiotic criteria and in particular for its ability to inhibit porcine pathogenic E. coli;
  • the Bacillus pumilus strain spore suspension administered has antimicrobial activity against E. coli in vitro due to the higher concentrations of propionic acid which is used to reduce the growth of Enterobacteriaceae; where intestinal concentrations of short chain fatty acid are increased by the Bacillus pumilus strain spore suspension as a result of carbohydrate degradation by the administered strains;
  • the Bacillus pumilus strain spore suspension lowered E. coli counts without toxic effects
  • liver weight was -10% decreased for animals fed the medicated treatment, which could be the result of chronic toxicity of apramycin leading to a reduction in size, when it is unable to regenerate following a period of ongoing insult.
  • the Bacillus pumilus strain spore suspension resulted in no histopathological abnormalities in the liver;
  • liver enzymes where compared to overall serum concentrations of the liver enzymes, GGT, ALT and
  • ALP were 21, 30.2 and 67.7% higher, respectively, in pigs fed the medicated treatment, indicating possible liver damage;
  • pigs fed the Bacillus pumilus strain spore suspension had lower percentages of lymphocytes and monocytes, compared to pigs on the non-medicated and medicated treatments, and to pigs on the non-medicated treatment, respectively;
  • a method for improving pig health comprising administering to pig a composition comprising a suspension of spores from a strain of Bacillus pumilus, or a variant thereof, and a pharmaceutically acceptable excipient.
  • a composition comprising a suspension of spores from a strain of Bacillus pumilus, or a variant thereof, and a pharmaceutically acceptable excipient.
  • the term "improving pig health” should be understood to mean decreased ileal E. coli counts, improved growth performance of the pig receiving the composition, improved liver function and physiology, improved renal function, reduced inflammation, improved feed conversion ratio when compared to pigs receiving medical treatment such as antibiotics, and/or improved intestinal tract physiology and beneficial gut flora.
  • composition comprising the B. pumilus strain is formulated for administration in a probiotic form.
  • the B. pumilus strain is a rifampicin-resistant strain isolated from seaweed.
  • the suspension of spores from the strain of Bacillus pumilus, or variant thereof has a concentration of 8 10 to 12 10 spores/ml, and ideally a concentration of 10 10 spores/ml.
  • the method comprises administering between 1 to 10 mis of the spore suspension.
  • the composition is administered orally.
  • the composition is administered daily.
  • the method comprises the step of administering the composition daily ad libitum.
  • the method comprises the step of administering initially at least 5 x 10 10 spores/ml to the pig in need thereof. Thereafter, the method comprises the step of administering 10 10 spores/ml to the pig in need thereof.
  • non-medicated diet negative control
  • medicated diet containing 200 mg apramycin/kg Apralan G200, Elanco Animal Health, Eli Lilly & Co., Basingstoke, Hampshire
  • pumilus WIT 588 daily (prepared and administered as outlined below). Treatments were administered continuously for 22 d post-weaning and pigs were provided with ad libitum access to feed and water. The diets were manufactured in the Moorepark feed mill and were formulated to meet or exceed the National Research Council requirements for weaned pigs. All phase 1 diets were formulated to 16.2 MJ/kg digestible energy and 16.2 g/kg lysine using the same ingredients except that apramycin and pharmacological levels of zinc oxide were added to the medicated diet.
  • phase 2 diets were formulated to 15.0 MJ/kg digestible energy and 15.0 g/kg lysine using the same ingredients except that apramycin and pharmacological levels of zinc oxide were added to the medicated diet. All diets were fed in 3 mm pellet form. Phase 1 diets were fed for 1 week after which phase 2 diets were fed for the remainder of the experiment.
  • Bacillus pumilus WIT 588 is a rifampicin resistant variant of a strain previously isolated from seaweed. It was generated to facilitate enumeration in the porcine GIT and characterized in vitro as a probiotic for animal production.
  • the strain was grown aerobically for -24 h in Brain Heart Infusion (BHI) broth (Oxoid Ltd, Basingstoke, Hampshire, UK) at 37 °C with agitation at 200 rpm. It was then induced to sporulate by spread-plating 1 mL of this culture onto sporulation agar and incubating for 7 d at 37 °C. The plates were then flooded with 10 mL sterile ice-cold water and the cells were suspended using a glass spreader. This suspension was heated at 80 °C for 15 min to kill any vegetative cells.
  • BHI Brain Heart Infusion
  • the spore concentration was determined by diluting the suspension 10-fold in maximum recovery diluent (MRD; Merck, Darmstadt, Germany), and spread-plating on BHI agar incubated at 37 °C for 24 h. The concentration was adjusted to ⁇ 10 10 spores/mL, and aliquots of this spore suspension were stored at -20 °C until use.
  • MRD maximum recovery diluent
  • Pigs were housed individually in fully slatted pens (1.2 m x 0.9 m) with plastic slats (Faroex, Manitoba, Canada) in a total of four rooms with 12 pigs per room. Each treatment group was represented in each room to avoid possible variation due to environment. The pigs had unlimited access to water from one nipple-in-bowl drinker (BALP, Charleville-Mezieres, Cedex, France) per pen. The temperature was controlled by a hot air heating system and an exhaust fan drawing air from under slat level, both controlled by a Stienen PCS 8400 controller (Stienen BV, Nederweert, The Netherlands).
  • ADFI average daily feed intake
  • ADG average daily gain
  • FCR feed conversion ratio
  • Blood samples were taken from each of 12 pigs/treatment by venipuncture from the anterior vena cava on d 0, 8 and 15. On d 22 of the experiment, 10 pigs/treatment were euthanized by captive bolt stunning followed by exsanguination and blood samples were taken at this time. All samples were collected in plastic blood collection tubes (Vacuette®, Labstock, Dublin, Ireland) and immediately inverted 10 times. Blood samples for serum biochemistry were collected in serum collection tubes, and allowed to clot at room temperature for 2-3 h prior to centrifugation (2000 x g for 10 min). Serum was collected and stored at -20 °C for subsequent biochemical analysis. Whole blood samples were collected in EDTA tubes and stored at room temperature for hematology analysis within 6 h of sampling.
  • EDTA blood samples were analyzed on a Beckman Coulter Ac T Diff (Beckman Coulter Inc., Brea, CA, USA), as outlined previously. Serum samples were analysed using an ABX Pentra 400 Clinical Chemistry Analyser (Horiba ABX, Northampton, UK), as outlined previously.
  • Samples of tissue were excised from two anatomical regions of the small intestine: the jejunum (55 cm distal to the pyloric junction) and the ileum (15 cm proximal to the ileo-cecal junction).
  • Samples of liver (centre of quadrate lobe) and kidney (cortex and medulla) were also taken and all samples were immediately placed in No-Tox fixative (Scientific Device Laboratory, Des Plaines, IL, USA) on a shaker for a minimum of 48 h.
  • Intestinal and organ samples were then treated, sliced and mounted and stained with haematoxylin and eosin (Sigma Aldrich, Ireland) for light microscopic examination. Determination of gross morphological parameters of the structure of the jejunum and ileum (villus height and crypt depth) was conducted. For each pig 10 villi and 10 crypts were measured on five fields of view, where villi were attached to the lumen. Measurements were taken from images obtained using a light microscope (Olympus, Southend-on-Sea, UK) fitted with an Optikam PR05 camera (Optika SRL, Ponteranica, Italy) using Optika- Vision Pro software.
  • the goblet cell number was determined in jejunal and ileal sections by periodic acid-Schiff staining. Positively stained periodic acid-Schiff cells were enumerated on 10 villi/sample. The means of all parameters were utilized for statistical analysis. All intestinal and organ tissue samples were also examined for histological evidence of abnormality by an experienced histopathologist.
  • Fecal samples were obtained by digital rectal stimulation from 12 pigs/treatment on d 0, 8, 15 and 20 and collected in sterile containers.
  • digesta samples from the cecum (terminal tip) and ileum (15 cm proximal to the ileo-cecal junction) were collected aseptically into sterile plastic containers. Both digesta and fecal samples were stored at 4 °C until analysis (within 12 h). Samples were homogenized and 500 ⁇ of each homogenate was heated to 80 °C for 15 min. Both heated and unheated homogenates were diluted.
  • Appropriate dilutions were plated, as follows; (1) unheated and heated samples were spread-plated on BHI agar containing 200 ⁇ g rifampicin/mL, 3.5% NaCl and 50 U/mL nystatin (Sigma) and incubated aerobically for 2 d at 37 °C to enumerate the vegetative cells + spores and spores alone, respectively of the administered B. pumilus strain; (2) unheated samples were pour-plated on ChromoCult® tryptone bile X- glucuronide (CTBX) agar (Merck) incubated at 44 °C for 24 h to enumerate E.
  • CTBX ChromoCult® tryptone bile X- glucuronide
  • D 0 values were used as a covariate in the model for analysis of hematology, serum gamma glutamyltransferase (GGT) and serum total protein (TP).
  • the appropriate covariance structure was fitted to the data.
  • the denominator degrees of freedom were computed using the Satterthwaite approximation. Fixed effects were treatment and sex. Block was included as a random effect. Simple main effects were obtained using the 'slice' option in SAS.
  • Least squares means were computed and P values were adjusted for multiple comparisons using the Tukey-Kramer adjustment. Significance was reported for P ⁇ 0.05 and tendencies towards significance were reported for 0.05 ⁇ P ⁇ 0.10. For all response criteria, the individual pig was considered the experimental unit.
  • No treatment x time interaction or treatment effect was observed for total white blood cell (WBC) counts (P > 0.05).
  • WBC white blood cell
  • Lymphocyte percentage was, however, lower for the B. pumilus treatment than for the other two treatments for the overall experimental period (P ⁇ 0.001) and at d 15 (P ⁇ 0.05). At d 8 the lymphocyte percentage was lower for the B.
  • pumilus treatment than the medicated treatment (P ⁇ 0.001), with the non-medicated treatment being similar to that of both other treatments (P > 0.05).
  • There was a treatment x time interaction for monocytes (%) (P 0.01). Monocyte percentage was higher for the non-medicated treatment than for the other two treatments for the overall period (P ⁇ 0.001) and at d 22 (P ⁇ 0.001).
  • the percentage of monocytes was higher for the non-medicated treatment than the B. pumilus treatment (P ⁇ 0.01), with that of the medicated treatment being similar to both other treatments (P > 0.05).
  • the percentage of granulocytes was higher for the B. pumilus treatment than for all other treatments for the overall period (P ⁇ 0.001), at d 8 (P ⁇ 0.001) and at d 15 (P ⁇ 0.01). At d 22, granulocyte percentage was higher for the B. pumilus treatment than for the non-medicated treatment (P ⁇ 0.01), with that of the medicated treatment being similar to that of both other treatments (P > 0.05).
  • MCH mean corpuscular hemoglobin
  • AST serum aspartate aminotransferase
  • Treatment did not affect renal or splenic weight; however, pigs on the medicated treatment had lighter livers than pigs on either the non-medicated or B. pumilus treatments (P ⁇ 0.05). Histopathological examination of the kidneys did not reveal any abnormalities for pigs on any of the treatments. Subtle inflammation was observed in the liver of one pig on the medicated treatment and in one pig on the B. pumilus treatment, but in both cases the inflammation was classified as 'very mild' in character and was most likely subclinical.
  • Treatment had no effect on crypt depth, villus width or villus height:crypt depth ratio in the jejunum.
  • the number of goblet cells/villus in the jejunum was higher for the B. pumilus and the medicated treatments compared to the non-medicated treatment (P ⁇ 0.01); however, the number of goblet cells/ ⁇ of villus was not affected by treatment (P > 0.05).
  • Treatment had no effect on any of the histological parameters investigated in the ileum. Histopathological examination revealed crypt inflammation in the ileum of one pig on the B. pumilus treatment, but this was categorized as very subtle and unlikely to be of clinical significance.
  • E. coli counts were lower for the medicated than both the non-medicated and B. pumilus treatments for the overall period (P ⁇ 0.01) as well as at d 8 (P ⁇ 0.001) and d 15 (P ⁇ 0.01).
  • none of the representative fecal E. coli isolates examined were hemolytic.
  • Lactobacillus counts were lower for the medicated than both the non-medicated and B. pumilus treatments for the overall period (P ⁇ 0.001) and at d 8 (P ⁇ 0.01).
  • Lactobacillus counts were lower for the medicated than the B. pumilus treatment (P ⁇ 0.01) while Lactobacillus counts for the non-medicated treatment were similar to those of both other treatments (P > 0.05).
  • the administered B. pumilus strain (vegetative cells plus spores as well as spores alone) was detected in the feces of all pigs on the B. pumilus treatment at all time points except d 0. The administered strain was not recovered from any of the pigs on either the non-medicated or medicated treatments throughout the experiment.
  • E. coli counts in the ileum were lower for the medicated and B. pumilus treatments compared to the non-medicated treatment (P ⁇ 0.05), but no treatment effect was observed for cecal E. coli counts (P > 0.05). However, none of the representative E. coli isolates recovered from either the ileum or cecum were hemolytic. Lactobacillus counts in the ileum were not affected by treatment (P > 0.05), while cecal Lactobacillus counts were lower for the medicated than the non- medicated and B. pumilus treatments (P ⁇ 0.05). The administered B.
  • pumilus strain (vegetative cells plus spores as well as spores alone) was detected in both the ileum and cecum of all pigs on the B. pumilus treatment but not from pigs on either the non-medicated or medicated treatments.
  • the pH of the ileal digesta was higher for pigs on the B. pumilus treatment compared to those on the non-medicated treatment (P ⁇ 0.05), with pigs on both treatments having ileal digesta with a similar pH to those on the medicated treatment (P > 0.05).
  • the pH of the cecal digesta was higher for pigs on the medicated treatment compared to those on the non-medicated and B. pumilus treatments (P ⁇ 0.01) with the latter two treatments having cecal content with a similar pH (P > 0.05).
  • the total concentration of short chain fatty acid in the ileum was higher for the medicated treatment than for the non-medicated treatment (P ⁇ 0.01) with that for the B. pumilus treatment being similar to that of both other treatments (P > 0.05). This was also the case for acetic acid concentrations in the ileum (P ⁇ 0.05).
  • the ileal concentration of propionic acid was similar for the medicated and B. pumilus treatments, but both treatments had higher concentrations than that found in the non-medicated treatment (P ⁇ 0.001).
  • P 0.08).

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Abstract

L'invention concerne une suspension de spores de souche de Bacillus pumilus destinée à être utilisée en tant que probiotique intégré aux aliments pour des animaux. La suspension répond à un certain nombre de critères pour probiotiques et en particulier quant à sa capacité à inhiber les bactéries E. coli pathogènes porcines. L'administration sous forme de supplément alimentaire de la suspension de spores de souche de Bacillus pumilus a amélioré les performances de croissance. La suspension a permis d'obtenir une quantité de bactéries E. coli iléale réduite ainsi qu'une amélioration de l'indice de consommation par comparaison avec un traitement médicamenteux du fait d'une augmentation du gain de poids journalier moyen. La survie de la suspension de spores de souche administrée dans les intestins a été démontrée. La diminution de la quantité de bactéries E. coli a été obtenue sans la réduction de la quantité de bactéries Lactobacillus potentiellement bénéfiques qui se produit avec les aliments médicamenteux.
PCT/EP2014/078428 2013-12-18 2014-12-18 Efficacité et innocuité d'une souche de bacillus d'origine marine pour une utilisation en tant que probiotique intégré aux aliments pour des cochons récemment sevrés WO2015091770A1 (fr)

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Publication number Priority date Publication date Assignee Title
EP3878980A1 (fr) * 2020-03-12 2021-09-15 Waterford Institute Of Technology Souche de bacillus altitudinis isolée et son utilisation en tant que probiotique

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3878980A1 (fr) * 2020-03-12 2021-09-15 Waterford Institute Of Technology Souche de bacillus altitudinis isolée et son utilisation en tant que probiotique
WO2021180970A1 (fr) * 2020-03-12 2021-09-16 Waterford Institute Of Technology Souche isolée de bacillus altitudinis et son utilisation en tant que probiotique

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